Protein tyrosine phosphorylation has been extensively characterized as a major mechanism of transducing signals within cells. The balance of tyrosine phosphorylation is maintained and modulated by two opposing sets of enzymes, the protein tyrosine kinases (PTKs) and the protein tyrosine phosphatases (PTPs). During embryonic development, several protein tyrosine kinases are known to have powerful and specific roles (Cantley et al., (1991) Cell 64:281-302; Fantl et al., (1993) Annu. Rev. Biochem. 62:453-481; Imamoto et al., (1994) Curr. Opin. Gen. Dev. 4:40-46; van der Geer et al., (1994) Annu. Rev. Cell Biol. 10:251-337). Though the PTPs are less well characterized, there is genetic evidence to indicate important functions in specific tissues during development. The Drosophila gene corkscrew, for example, encoding an intracellular PTP, is required for the development of the head and tail of the embryo (Perkins et al., (1992) Cell 70:225-236). Mice homozygous for the moth-eaten (me) allele which encodes a mutated version of the intracellular PTP, hematopoietic cell phosphatase (HCP, also known as SH-PTP1 and PTP1C), have a variety of defects in the immune system (Shultz et al., (1993) Cell 73:1445-1454; Tsui et al., (1993) Nat. Gen. 4:124-129). Important roles for other PTPs are also indicated by biochemical studies. For instance, an intracellular PTP, FAP-1 (also known as PTP-BAS) has been found to be involved in the signal transduction pathway of apoptosis (Sato et al., (1995) Science 268:411-415).
In addition to the phosphatase catalytic domain, many PTPs contain a transmembrane and extracellular domain (Cohen and Cohen, (1989) J. Biol. Chem. 264:21345-21438; Hunter, (1989) Cell 58:1013-1016; Walton and Dixon, (1993) Annu. Rev. Biochem. 62:101-120; Brady-Kalnay and Tonks, (1995) Curr. Opin. Cell Biol. 7:650-657). Like the transmembrane PTKs, the transmembrane PTPs could be receptors with the potential to regulate the phosphorylation state of downstream targets in response to binding of extracellular ligands. However, there has heretofore been little evidence on ligands, or on the potential for ligand-induced signaling. Two transmembrane PTPs, PTPm and PTPk, have been demonstrated to exhibit homophilic binding which can cause cell-cell adhesion (Brady-Kalnay et al., (1993) Curr. Opin. Cell Biol. 7:650-657; Gebbink et al., (1993) J. Biol. Chem. 268:16101-16104; Sap et al., (1994) Mol. Cell. Biol. 14:1-9). Another transmembrane PTP, RPTPb, was found to correspond to phosphacan, a proteoglycan that can interact with the adhesion molecules N-CAM and Ng-CAM and the extracellular matrix protein tenascin (Milev et al., (1991) J. Cell Biol. 127:1703-1715; Barnea et al., (1994) J. Biol. Chem. 269:14349-14352; Grumet et al., (1994) J. Biol. Chem. 269:12142-12146), and was also identified as a ligand of the neuronal cell surface molecule contactin (Peles et al., (1995) Cell 82:251-260).
In vertebrates and invertebrates, several receptor-type PTPs have been identified with restricted expression patterns in the developing nervous system. In Drosophila, DPTP99A, DPTP10D, and DLAR are transmembrane PTPs with neuron-specific expression, and immunolocalization of these molecules on axons has led to proposals that they may be involved in axon outgrowth and guidance (Tian et al., (1991) Cell 67:687-700; Yang et al., (1991) Cell 67:661-673). In vertebrates, RPTPb shows expression restricted to the developing nervous system (Carnoll et al., (1993) Brain Res. Dev. Brain Res. 75:293-298; Levy et al., (1993) J. Biol. Chem. 268:10573-10581). LAR, RPTPs ,and CRYPa were also found expressed in embryonic neuronal tissues (Yan et al., (1993) J. Biol. Chem. 268:24880-24886; Stoker (1994) Mech. Dev. 46:201-217; Wang et al., (1995) J. Neurosci. Res. 41:297-310). In addition, PTPa was found to be induced during neuronal differentiation of P 19 cells (den Hertog et al., (1993) EMBO J. 12:3789-3798). Similarly, the expression of two other PTPs, PC12-PTP1 and LAR, was induced in differentiating PC12 cells (Sharama and Lombroso, (1995) J. Biol. Chem. 270:49-53; Zhang and Longo, (1995) J. Cell. Biol. 128:415-431).
Unlike the nervous system, there is little information on molecular mechanisms for cell-cell signaling in pancreatic development, and no cell-cell signaling molecules specific to the pancreatic lineage have yet been identified. The pancreas is of enormous medical importance, because of its role in widespread diseases, notably juvenile diabetes and pancreatic cancer. Formation of the pancreas during development has been well studied at the morphological and cellular level, but little is known about control of the induction, growth or differentiation of the pancreas at the molecular level (Slack, (1995) Development 121:1569-1580). Previous studies have shown some transcription factors expressed in early developing pancreas. In particular, STF-1 (also known as IPF-1, IDX-1, or PDX: Ohlsson et al., (1993) EMBO J. 12:4251-4259; Miller et al., (1994) EMBO J. 13:1145-1156; and Guz et al., (1995) Development 121:11-18), a homeobox gene, is expressed in the pancreatic primordium and adjacent gut endothelium, and has been shown by targeted mutagenesis to be critical for the development of the pancreas (Jonsson et al., (1994) Development 114:75-87). However, even though extracellular signals to control pancreatic endocrine development could be clinically useful, the mechanisms of extracellular signaling that control pancreas formation and endocrine cell development are still being elucidated.
The present invention relates to the discovery of a new class of the receptor protein tyrosine phosphatases (PTP), referred to herein as PTP-NP (for neural and pancreatic) receptors.
In general, the invention features isolated PTP-NP polypeptides, preferably substantially pure preparations of the subject PTP-NP polypeptides. The invention also provides recombinantly produced PTP-NP polypeptides. In preferred embodiments the polypeptide has a biological activity including one or more of: the ability to dephosphorylate a phosphotyrosine residue; hydrolyze a phosphatase substrate such as p-nitrophenylphosphate; bind to a ligand expressed on pancreatic xcex2 cells. However, PTP-NP polypeptides which specifically antagonize such activities, such as may be provided by truncation mutants or other dominant negative mutants, are also specifically contemplated.
The PTP-NP proteins of the present invention can be characterized as including one or more of the following domains/motifs: an extracellular domain, having a cys4 domain, which mediate ligand binding, a transmembrane domain, and an intracellular domain including a phosphatase domain. The protein may also include a secretion signal sequence, and (optionally) glycosylated amino acid residues.
In one embodiment, the polypeptide is identical with or homologous to a PTP-NP protein represented in SEQ ID NO: 2. Related members of the PTP-NP family are also contemplated, for instance, a PTP-NP polypeptide preferably has an amino acid sequence at least 65%, 70%, 75% or 80% homologous to the polypeptide represented by. SEQ ID NO: 2, though polypeptides with higher sequence homologies of, for example, 85, 90% and 95% or are also contemplated. In a preferred embodiment, the PTP-NP polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions with a nucleic acid sequence represented in SEQ ID NO: 1. Homologs of the subject PTP-NP proteins also include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with extracellular ligands or with intracellular proteins involved in signal transduction.
The PTP-NP polypeptide can comprise a full length protein, such as represented in SEQ ID NO: 2, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length. In preferred embodiments, the PTP-NP polypeptide includes a sufficient portion of the extracellular domain to be able to specifically bind to a PTP-NP ligand. Truncated forms of the protein include, but are not limited to, soluble extracellular domain fragments including the CYS4 motif, soluble intracellular domains including the phosphatase domain, and membrane-bound forms of either which include the transmembrane domain. Another preferred fragment includes at least 5, though more preferably at least 10, 20, 30 or more residues N-terminal to proline 282 of SEQ ID NO: 2.
In certain preferred embodiments, the invention features a purified or recombinant PTP-NP polypeptide having a core polypeptide molecular weight of about 111.5 kd including a signal sequence, e.g., in the range of 105 kd to 115 kd. In other embodiments, the peptide core of a mature PTP-NP protein preferably has a molecular weight of about 108.8 kD. It will be understood that certain post-translational modifications, e.g., glycosylation and the like, can increase the apparent molecular weight of the PTP-NP protein relative to the unmodified polypeptide chain.
The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the PTP-NP protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the PTP-NP polypeptide, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e.g. the second polypeptide portion is an epitope tag.
In yet another embodiment, the invention features a nucleic acid encoding a PTP-NP polypeptide, which has the ability to modulate, e.g., either mimic or antagonize, at least a portion of the activity of a wild-type PTP-NP polypeptide. An exemplary PTP-NP-encoding nucleic acid sequence is represented by SEQ ID NO: 1.
In another embodiment, the nucleic acid of the present invention includes a coding sequence which hybridizes under stringent conditions with the coding sequence designated in SEQ ID NO: 1. The coding sequence of the nucleic acid can comprise a sequence which is identical to a coding sequence represented in of SEQ ID NO: 1, or it can merely be homologous to that sequences. In preferred embodiments, the nucleic acid encodes a polypeptide which specifically modulates, by acting as either an agonist or antagonist, one or more of the bioactivities of a wild-type PTP-NP polypeptides.
Furthermore, in certain preferred embodiments, the subject PTP-NP nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the PTP-NP gene sequence. Such regulatory sequences can be used in to render the PTP-NP gene sequence suitable for use as an expression vector. This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing PTP-NP proteins by employing said expression vectors.
In yet another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of either sense or antisense sequence of SEQ ID NO: 1; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of SEQ ID NO: 1.
Yet another aspect of the present invention concerns an immunogen comprising a PTP-NP polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a PTP-NP polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from the protein represented by SEQ ID NO: 2.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the PTP-NP immunogen.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, hating a transgene, e.g., animals which include (and preferably express) a heterologous form of a PTP-NP gene described herein, or which misexpress an endogenous PTP-NP gene, e.g., an animal in which expression of one or more of the subject PTP-NP proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed PTP-NP alleles or for use in drug screening.
The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequence of SEQ ID NO: 1, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a PTP-NP protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a PTP-NP protein; e.g. measuring a PTP-NP mRNA level in a cell, or determining whether a genomic PTP-NP gene has been mutated or deleted. These so-called xe2x80x9cprobes/primersxe2x80x9d of the invention can also be used as a part of xe2x80x9cantisensexe2x80x9d therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject PTP-NP proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25, 40, 50, or 75 nucleotides in length are also contemplated.
In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a PTP-NP protein and, for example, a virus, an extracellular ligand of the PTP-NP protein, or an intracellular protein which binds to the PTP-NP protein, e.g., a substrate of the PTP-NP phosphatase activity. An exemplary method includes the steps of (i) combining a PTP-NP polypeptide or bioactive fragments thereof, a PTP-NP target molecule (such as a PTP-NP ligand or a PTP-NP substrate), and a test compound, e.g., under conditions wherein, but for the test compound, the PTP-NP protein and target molecule are able to interact; and (ii) detecting the formation of a complex which includes the PTP-NP protein and the target polypeptide either by directly quantitating the complex, by measuring inductive effects of the PTP-NP protein, or, in the instance of a substrate, measuring the conversion to product. A statistically significant change, such as a decrease, in the interaction of the PTP-NP and target molecule in the presence of a test compound (relative to what is detected in the absence of the test compound) is indicative of a modulation, e.g., inhibition or potentiation, of the interaction between the PTP-NP protein and the target molecule.
Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a cell by modulating PTP-NP bioactivity, e.g., by potentiating or disrupting certain protein-protein interactions. In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a PTP-NP therapeutic so as to alter, relative to the cell in the absence of treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with PTP-NP therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a PTP-NP protein or ligand binding of a PTP-NP protein. Other PTP-NP therapeutics include antisense constructs for inhibiting expression of PTP-NP proteins, and dominant negative mutants of PTP-NP proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type PTP-NP protein.
Another aspect of the present invention provides a method of determining if a subject, e.g. an animal patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation or apoptosis. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a PTP-NP protein, e.g. represented in SEQ ID NO: 1 or a homolog thereof; or (ii) the mis-expression of a PTP-NP gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a PTP-NP gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble PTP-NP protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a PTP-NP gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the PTP-NP gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the PTP-NP gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a PTP-NP protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the PTP-NP protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.